US7803902B2ActiveUtilityPatentIndex 72
Modified variant bowman birk protease inhibitors

Assignee: DANISCO US INCPriority: Oct 15, 2008Filed: Oct 15, 2008Granted: Sep 28, 2010
Est. expiryOct 15, 2028(~2.3 yrs left)· nominal 20-yr term from priority
Inventors:AMIN NEELAM S COLLIER KATHERINE D ESTABROOK MELODIE ESTELL DAVID A FOX BRYAN P POWER SCOTT D SCHMIDT BRIAN F VOGTENTANZ GUDRUN
C07K 14/811A61K 8/64A61Q 7/02
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Cited by
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References
Claims
Abstract

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.
Claims

exact text as granted — not AI-modified
1. An isolated modified variant Bowman Birk Protease Inhibitor (BBPI) comprising
 a BBPI scaffold selected from the group consisting of: BBI (SEQ ID NO: 13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454),
 wherein the second protease inhibitory loop of the BBPI scaffold is replaced with a binding peptide selected from the group consisting of a vascular endothelial growth factor (VEGF) binding peptide, a fibroblast growth factor-5 (FGF-5) binding peptide, a transforming growth factor β (TGFβ) binding peptide and a tumor necrosis factor α (TNFα) binding peptide; and 
 wherein the backbone of the BBPI scaffold is substituted at one or more amino acid positions selected from the group consisting of positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187;
 wherein the substitution at position 1 is to A or C, the substitution at position 4 is to V, the substitution at position 5 is to P or A, the substitution at position 11 is to G, the substitution at position 13 is to Y, I, F, M, L, V, K or R, the substitution at position 18 is to I, V or L, the substitution at position 25 is to K, N, W, I, A, R or L, the substitution at position 27 is to R, K, V, A or Q, the substitution at position 29 is to R, K, P or E, the substitution at position 31 is to Q, H, E, A, R, W, K or T, the substitution at position 38 is to N, K or R, the substitution at position 40 is to H, K, Q, R or Y, the substitution at position 50 is to R, Q, K, T, V, M or S, the substitution at position 52 is to K, T, R, Q, L, H, A, M, S or E, the substitution at position 55 is to M and the substitution at position 65 is to E, Q or D. 
 
 
 
     
     
       2. The isolated modified variant BBPI of  claim 1 , wherein said binding peptide is a VEGF binding peptide chosen from YNLYGWT (SEQ ID NO: 676), ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460),SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), HLAPSAV (SEQ ID NO:519), KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO: 531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536). 
     
     
       3. The isolated modified variant BBPI of  claim 1 , wherein said binding peptide is an FGF-5 binding peptide chosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671). 
     
     
       4. The isolated modified variant BBPI of  claim 1 , wherein said binding peptide is a TGFβ binding peptide chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO:672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674). 
     
     
       5. The isolated modified variant BBPI of  claim 1 , wherein said binding peptide is a TNFα binding peptide chosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594). 
     
     
       6. The isolated modified variant BBPI of  claim 1 , wherein said modified variant BBPI is expressed as a fusion protein comprising a catalytic domain chosen from cellulase, cutinase and disulfide isomerase. 
     
     
       7. The isolated modified variant BBPI of  claim 1 , wherein said modified variant BBPI is expressed as a fusion protein consisting of SEQ ID NO:195. 
     
     
       8. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of two amino acid positions equivalent to positions 50 and 52 of SEQ ID NO:187. 
     
     
       9. The isolated modified variant BBPI of  claim 8 , wherein said modified variant BBPI comprises SEQ ID NO:595. 
     
     
       10. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of three amino acid positions chosen from amino acid positions equivalent to positions 25, 29, 40, 50 and 52 of SEQ ID NO:187. 
     
     
       11. The isolated modified variant BBPI of  claim 10 , wherein said combination is chosen from 25L-50T-52A, 29P-50T-52A, and 40K-50T-52A. 
     
     
       12. The isolated modified variant BBPI of  claim 10 , wherein said modified variant BBPI comprises SEQ ID NO:603, 607 or 609. 
     
     
       13. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of four amino acid positions chosen from amino acid positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. 
     
     
       14. The isolated modified BBPI of  claim 13 , wherein said combination is chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-40K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, 29P-40K-50T-52A and 13I-25K-29P-52K. 
     
     
       15. The isolated modified variant BBPI of  claim 14 , wherein said modified variant BBPI comprises SEQ ID NO: 432, 434 443, 445, 447, 596, 600, 602, 604, 606, 608, or 643. 
     
     
       16. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of five amino acid positions selected from amino acid positions equivalent to positions 13, 25, 29, 40, 50, and 52 of SEQ ID NO:187. 
     
     
       17. The isolated modified variant BBPI of  claim 16 , wherein said combination is chosen from 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, 13I-29P-40K-50T-52A, 25L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13I-29P-40K-50T-52T. 
     
     
       18. The isolated modified variant BBPI of  claim 17 , wherein said modified variant BBPI comprises SEQ ID NO: 597, 599, 601, 605, 615, 620, 624, or 625. 
     
     
       19. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of six amino acid positions chosen from amino acid positions equivalent to positions 1, 4, 5, 11, 13, 25, 27, 29, 31, 38, 40, 50, 52 and 65 of SEQ ID NO:187. 
     
     
       20. The isolated modified variant BBPI of  claim 19 , wherein said combination is chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-40K-50T-52A-65E. 
     
     
       21. The isolated modified variant BBPI of  claim 20 , wherein said modified variant BBPI comprises SEQ ID NOS:598, 611, 612, 613, 614, 616, 619, 621, 622, 623, or 626. 
     
     
       22. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of seven amino acid positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. 
     
     
       23. The isolated modified variant BBPI of  claim 22 , wherein said combination is chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A and 13I-25R-27A-29P-31A-50K-52T. 
     
     
       24. The isolated modified variant BBPI of  claim 22 , wherein said modified variant BBPI comprises SEQ ID NOS: 491, 617, 618, 632, 633, 634, 635, 636, 637, 638 or 639. 
     
     
       25. The isolated modified variant BBPI of  claim 1 , wherein the backbone of the BBPI scaffold is substituted at a combination of eight amino acid positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. 
     
     
       26. The isolated modified BBPI of  claim 25 , wherein said combination is chosen from 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L and 13I-25K-27Q-29P-31E-40H-50R-52Q. 
     
     
       27. The isolated modified variant BBPI of  claim 25 , wherein said modified variant BBPI comprises SEQ ID NO: 627, 628, 629, 630 or 631. 
     
     
       28. The isolated modified variant BBPI of  claim 1 , wherein said modified variant BBPI binds VEGF. 
     
     
       29. The isolated modified variant BBPI of  claim 1 , wherein said VEGF binding peptide is chosen from SEQ ID NOS: 676, 9, 458, 459, 460, 468, 469, 470, 471, 472 and 473. 
     
     
       30. The isolated modified variant BBPI of  claim 1 , wherein said modified variant BBPI binds VEGF and comprises SEQ ID NO: 601, 602, 627, 628, 630, 631, 643, 491, 632, 633, 634, 635, or 636. 
     
     
       31. The isolated modified variant BBPI of  claim 1 , wherein the substitutions in the backbone of the BBPI scaffold increase the trypsin inhibitory activity of the modified variant BBPI. 
     
     
       32. The isolated modified variant BBPI of  claim 1 , wherein the substitutions in the backbone of the BBPI scaffold increase the trypsin inhibitory activity and production yield of the modified variant BBPI. 
     
     
       33. An isolated modified variant Bowman Birk Protease Inhibitor (BBPI) comprising
 a BBPI scaffold consisting of BBIt-AV (SEQ ID NO:187),
 wherein the VEGF binding peptide of the BBPI scaffold is replaced with a binding peptide selected from the group consisting of a FGF-5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide; and 
 wherein the backbone of the BBPI scaffold is substituted at one or more amino acid positions selected from the group consisting of 13, 25, 27, 29, 31,40, 50 and 52;
 wherein the substitution at position 13 is to Y, I, F, M, L, V, K or R, the substitution at position 25 is to K, N, W, I, A or R, the substitution at position 27 is to R, K, V, A or Q, the substitution at position 29 is to R, K or P, the substitution at position 31 is to Q, H, E, A, R, W, K or T, the substitution at position 40 is to H, K, Q, R or Y, the substitution at position 50 is to R, Q, K, T, V, M or S, the substitution at position 52 is to K, T, R, Q, L, H, A, M, S or E. 
 
 
 
     
     
       34. The isolated modified variant BBPI of  claim 33 , wherein said binding peptide is an FGF binding peptide chosen from SEQ ID NOS: 430, 431, 670, and 671. 
     
     
       35. The isolated modified variant BBPI of  claim 33 , wherein said binding peptide is a TGF binding peptide chosen from SEQ ID NOS: 436, 437, 438, 672, 673, and 674. 
     
     
       36. The isolated modified variant BBPI of  claim 33 , wherein said binding peptide is a TNF binding peptide chosen from SEQ ID NOS: 474, 475, and 476. 
     
     
       37. The isolated modified variant BBPI of  claim 33 , wherein the substitutions in the backbone of the BBPI scaffold increase the trypsin inhibitory activity and production yield of the modified variant BBPI. 
     
     
       38. The isolated modified variant BBPI of  claim 33 , wherein said modified variant BBPI comprises SEQ ID NO: 432, 434, 443, 445, 447, 637, 638, and 639.
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